Tropical and Subtropical Agroecosystems 23 (2020): #48 Holguin-Peña et al., 2020

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PLANT GROWTH PROMOTING BACTERIA AND

MYCORRHIZAL ON VARIETIES OF Sorghum spp.

GERMINATION UNDER STRESSING ABIOTIC CONDITIONS †

[BACTERIAS PROMOTORAS DEL CRECIMIENTO VEGETAL

Y MICORRIZAS SOBRE LA GERMINACION EN VARIEDADES

DE Sorghum spp. BAJO CONDICIONES DE ESTRESES

ABIÓTICOS]

Ramón Jaime Holguin Peña1, Juan Manuel Vargas López2,

Guadalupe Amanda López Ahumada2, Francisco Rodríguez Félix2,

Alberto Olguín Moreno2, Carlos Borbón Morales3,

Juan Pedro López Córdova4 and Edgar Omar Rueda-Puente4*

1 Centro de Investigaciones Biológicas del Noroeste, Mar Bermejo No.

195, Col. Playa Palo Santa Rita, La Paz, Baja California Sur CP 23090. 2 Universidad de Sonora, Departamento de Investigación y Posgrado en

Alimentos, Colonia centro Hermosillo, Sonora, México CP 83000. 3 Centro de Investigación en Alimentación y Desarrollo AC,

Coordinación de Desarrollo Regional, Departamento de Economía.

Carretera a la Victoria Km. 0.6, Sonora, México. CP 83304. 4 Universidad de Sonora, Departamento de Agricultura y Ganadería,

Carretera a Bahía de Kino, km 20.5, Hermosillo, Sonora, México, 83000.

Email. erueda04@santana.uson.mx

* Corresponding author

SUMMARY

Background. Mexico currently ranks first in Sorghum production. Notwithstanding, soils in a productive

area of Sonora are too poor in organic matter with saline intrusion and high temperatures. Farmers apply

synthetic fertilizers but the indiscriminate use increase salinity. Under dry and arid zones, plant growth

promoting bacteria (PGPB) and arbuscular mycorrhizal fungi (AMF) signifies a substitute to obtain

nutrients. Objective. New and productive varieties of Sorghum in Sonora desert (Silo Miel 370 VCB (370),

Sorgo Silo Miel 315 VC (315), Sorgo Silo Miel II (II) and Camino 526 Croplan winfield (526)) were

evaluated inoculated with a species PGPB and AMF. Methodology. Under salinity, temperature regime

(night/ day) in germination stage, tests were carried out considering the inoculation with PGPB and AMF;

growth variables were measured like a percentage and rate, plant height, root length, and produced biomass

(fresh and dry matter). Results. The cultivar 526 was the most outstanding with the PGPB and AMF

evaluated; at the seedling stage, there were significant differences between the treatments for plant height,

root length, fresh and dry weight, considering NaCl levels and temperature regimes. Implication. Studies

of the association of PGPB and AMF with the Sorghum cv. 526 are recommended under field conditions.

Conclusion. This study is the first step to obtain an ideal variety of Sorghum, which is a principal crop in

dry arid and desert zones of México. This kind of study promotes beneficial microorganisms as a resource

to increase the development and productivity of plants.

Keywords: temperature; salinity; basic crops.

RESUMEN

Antecedentes. Actualmente, México ocupa el primer lugar en producción de sorgo. No obstante, los suelos

en un área productiva de Sonora son demasiado pobres en materia orgánica con intrusión salina y altas

temperaturas. Los agricultores aplican fertilizantes sintéticos, pero el uso indiscriminado aumenta la

salinidad. En zonas áridas, las bacterias promotoras del crecimiento vegetal (PGPB) y los hongos

micorrícicos arbusculares (AMF) representan un sustituto para obtener nutrientes en los cultivos. Objetivo.

Se evaluaron las variedades nuevas y productivas del desierto de Sonora (Silo Miel 370 VCB (370), Sorgo

Silo Miel 315 VC (315), Sorgo Silo Miel II (II) y Camino 526 Croplan winfield (526)) al inocularse con

una especie de PGPB y AMF. Metodología. Bajo condiciones de salinidad, un régimen de temperatura

(noche / día) en la etapa de germinación, se realizaron pruebas considerando la inoculación con la PGPB y

† Submitted August 27, 2019 – Accepted January 6, 2020. This work is licensed under a CC-BY 4.0 International License.

ISSN: 1870-0462.

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el AMF; las variables de crecimiento se midieron como porcentaje y tasa de germinación, altura de la

plántula, longitud de la raíz y biomasa producida (materia fresca y seca). Resultados. El cultivar 526 fue

el más sobresaliente con la PGPB y el AMF evaluados; en la etapa de plántula existieron diferencias

significativas entre los tratamientos para la altura de la planta, longitud de la raíz, el peso fresco y seco

considerado los niveles de NaCl y los regímenes de temperatura. Implicaciones. Estudios de la asociación

de PGPB y AMF con el sorgo var. 526 se recomiendan en condiciones de campo. Conclusión. Este estudio

es el primer paso para obtener una variedad ideal de sorgo, que es un cultivo principal en zonas secas, áridas

y desérticas de México. Este tipo de estudio promueve microorganismos benéficos como un recurso para

aumentar el desarrollo y la productividad de las plantas.

Palabras clave: temperatura; salinidad; cultivo básico.

INTRODUCTION

Sorghum (Sorghum bicolor) is one principal

cereal crop in many countries; it contains high

levels of minerals and vitamins, i.e. those of

group B are listed thiamine, riboflavin, and

niacin (López-Ortíz et al. 2011). This richness

makes it an important antioxidant food. While it

is also rich in iron, calcium, phosphorus, zinc and

insoluble fiber (de Morais et al. 2017). Sorghum

(subsp. bicolor) belongs to the family Gramineae

and it is sowed in countries of America (Mexico,

Argentina, and the USA), Africa (where the

Sorghum is originated), Asia and Oceania

(Ethiopia, Sudan; Chine; Australia,

respectively). Sorghum is a protein-rich cereal

and stands out for containing a lower caloric

intake, with fewer carbohydrates and fats.

Besides, it is a gluten-free food, so it is optimal

for celiac people. Besides, it has antidiarrheal and

homeostatic properties. Currently, new markets

for Sorghum are developed producing novel

functional foods. Mexico currently ranks first in

Sorghum production with 11 million tons per

year. Likewise, our country has reached the

world record in yield of Sorghum of up to 17 tons

per hectare, reflecting that this crop is one of the

most productive crops.

Sonora and Baja California Peninsula are some

of the aridest states of Mexico, with 80 mm

average annual precipitation (Rueda et al. 2004).

The main problems facing agriculture are a

declining supply of water and a decrease in the

quality of the water. While the total world

population today demands proportionally more

and more food, that total is expected to exceed

9,000 million by 2050. In the agricultural sector,

many adverse factors reduce productivity, i.e.

water extraction in excess, use of inappropriate

fertilizers, which have caused increases in the

salinity of agriculture soil (SAGAR 1981; Salim

1989; Shalaby et al. 1993; Sangakkara et al.

1995; Abdullah et al. 2001; Rashid et al. 1999).

In this sense in dry arid and desert zones, Novo

production alternatives are developing

concerning the selection and evaluation of salt-

tolerant plants that already are adapted to salty

areas (Flowers and Yao 1995; Flowers 2004).

Studies with a salt-tolerant plant have been

carried out in an agronomic, physiological,

biochemical, and molecular sense. However, in

recent years, the number of works that address

tolerance to salinity from a germinations stage,

point of view has increased considerably, to

search responses under different conditions,

because the variability is extensive (Abdus et al.

1999; Acosta-Motos et al. 2017; Miranda et al.

2017; Chen et al. 2018; Yan et al. 2018; Dilnur

et al. 2019; Hernández 2019; Zhong et al. 2019).

However, although there are many Sorghum

varieties with a high viable seed, the germination

and establishment are poor under the soils of the

Sonora region (Zhang et al. 2019). In this sense,

farmers due the climatic conditions are favorable

to produce Sorghum, they invest in agro-inputs

and technology, so in the Mexican Sonora state,

the Sorghum has a wide distribution along with

the central parts of Sonora state (Zhang et al.

2019).

Moreover, the productivity of Sorghum is limited

by the absence of accessible nitrogen (de Morais

et al. 2017; SAGAR 1981). This situation

disturbs its development and reproduction

possible (Juarez and Harrison 2018).

Traditionally, growers apply artificial fertilizers

to recompense for soil nitrogen deficit. However,

unselective use of these nourishments forces the

increasing salinity and severely injuries soil

microorganism's structure and configuration

(Jefferies 1977; Kapulnik et al. 1981; Banwari

and Rao 1990; Nahid and Gomah 1991).

Few studies on plant growth-promoting bacteria

(PGPB) have been published of Sorghum spp

(Akhavan et al. 1991; Isopi et al. 1995; Luigi et

al. 1998; Compant et al. 2010), but not in

association with mycorrhizas diversity within the

rhizosphere. Concerning varieties sowed in the

Sonora desert, the studies are null. So is very

important to increase the number of known salt-

tolerant, nitrogen-fixing bacteria and arbuscular

mycorrhizal (Hamdi 1999; Whipps 2000).

On the other hand, several principal stressing

factors as a temperature (Adams et al. 2001;

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Aksouh et al. 2006; Luo 2011), and salinity

(Pichu 2010; Kroes and Supit 2011) has been

proved in glicophytes, which regulate some

stages of the plants (germination, pre-flowering,

flowering and mature physiologic) and interact

within the soil interface (Ungar 1995; Laynez-

Garsaball et al. 2007). The osmotic and matrix

potential in saline soils decreases the temperature

that is effective for seed germination (Hegarty

1978). The adverse factor such as salinity

interacting with another abiotic factor such as

temperature, determine high germination

percentages (Khan and Ungar 1998; Rivers and

Weber 1971; Philipupiya and Ungar 1984;

Keiffer and Ungar 1995; Khan and Ungar 1996;

Khan and Ungar 1998; Gulzar et al. 2000); it is

worth mentioning that, the ability of plants to

counteract adverse factors is conditioned by

multiple biochemical and physiological

pathways that help to better capture cellular

water; physiological protection of chloroplasts,

maintaining a homeostasis of the ions. Plants to

detoxify radicals under salinity conditions

activate to essential routes such as the synthesis

of osmotically active metabolites, specific

proteins and certain free radical elimination

enzymes that maintain a balance and flow of ions

and water; they also favor and contribute a

function in the elimination of oxygen radicals or

chaperones; some species accumulate

methylated metabolites, whose function is

osmoprotectant and eliminate free radicals.

Photorespiration is a physiological phenomenon

which it is altered under abiotic conditions (Asish

and Anath 2005). In the case of halophyte seeds,

they can be viable for prolonged periods when

they are in hypersaline conditions, allowing them

to germinate when the conditions of salinity in

the soil and water are reduced (Khan et al. 1976;

Woodell 1985; Khan and Ungar 1997; Khan and

Ungar 1999).

Notwithstanding the foregoing, it is important to

indicate that among salinity tolerant plants, there

is variability to respond positively and negatively

to adverse conditions varying conditions such as

temperature and salinity (Khan and Ungar 1997;

Gul and Weber 1999). The present study defines

the influence of the inoculation with the PGPB

(Bacillus amyloliquefaciens) and the AMF

(Glomus intraradices) under different conditions

of salinity and discontinuous temperature

regimes on the seed germination of varieties of

Sorghum bicolor.

MATERIALS AND METHODS

Effect on germination and seedling growth of

four varieties of Sorghum spp. by inoculants

in NaCl conditions

Seeds of four varieties of Sorghum spp. were

collected, from production areas of Sonora,

Northwest of Mexico. The collected varieties

were Silo Miel 370 VCB (370), Sorgo Silo Miel

315 VC (315), Sorgo Silo Miel II (II) and

Camino 526 CROPLAN WINFIELD (526).

Largest and health seeds were carefully chosen

and with no observable destruction. Seeds of

each variety were disinfected in sodium

hypochlorite (3% active chlorine) for 10 s, and

washed with sterilized and distilled water and

dried with sterile paper. The halo bacterium B.

amyloliquefaciens (Accession number

KJ433614), on N-free OAB agar medium

according to (Renganathan et al. 2019), with

three different concentrations of NaCl (0.25 M)

was replicated. Later, at a concentration of 108

cells.mL-1, seeds were inoculated with bacteria

treatments according to (Carrillo et al. 1998). At

the same time and according to (Strullu and

Romand 1986; Declerck et al. 1998; Bécard and

Piché 1992; Bécard and Fortin 1988), and using

Allium schoenoprasum was reproduced Glomus

intraradices. The inoculum obtained was from a

sample of AM 120 Granite Seed Company (Lehi,

UT 84043). Furthermore, seeds were treated with

the AMF-G. intraradices according to (Rueda-

Puente et al. 2018), using a concentration of 120

propagules.cm3. Into the sterilized Petri dishes,

germination tests were performed, each Petri

dish with a cotton layer substrate (150 x 15 mm)

covering the bottom of the dish. Dishes were

moistened with uniform NaCl solution (0, 0.06,

or 0.12 M). All seeds varieties considering its

treatment and separated form were done inside a

growth chamber at 27°C ± 0.5°C and 35% ± 1%

RH, with continuous white light. Every four

days, 20 mL of the appropriate solution was

added to each dish. When the radicle was at least

2 mm long from seeds, were considered

germinated. Final percentage germination was

measured after 25 days; also the number of

germinated seeds was recorded daily

(germination rate). Using the next formula, the

germination rate was calculated (Maguire 1962):

M=n1/t1 + n2/t2 +…n25/t25

where n1, n2, … n25 are the numbers of

germinated seeds at times t1, t2, … t25 in days.

The hierarchic experiment of a randomized

design included three factors (varieties,

inoculation, and salinity), with five replicates of

50 seeds. The first factor (variety) had four levels

(varieties: 370, 315, II and 526); three

bioinnoculations: without inoculation,

inoculation with mycorrhiza arbuscular (Glomus

intraradices) and inoculation with the bacterium

Bacillus amyloliquefaciens were the second

factor; and the three concentrations of NaCl (0,

0.06, and 0.12 M) were the third factor. The three

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studied factors combination turned out 36

treatments. Arcsine transformation was realized

to obtain percentage germination (56), with

three-way analyses of variance (ANOVA).

Germination rates were obtained by sums of

germinations per day and were not transformed

before analysis. Duncan’s multiple range test at

P = 0.05 was carried out to obtain significant

differences between means of treatments. Data

were analyzed using the Statistical Analysis

System (Sokal and James 1962). Seedling

growth of four varieties under all conditions was

measured by dry and fresh weights on 30 days

after inoculation (dai); other variables calculated

were root length and height and dry weight.

Colony-forming units (CFU) was a measurement

in the root system from seedlings considering

Carrillo et al. (Bashan et al. 2007). In all

variables least significant differences (LSD)

were obtained by Duncan’s Multiple Range Test

at P = 0.05. All statistical analysis was done with

SAS (2004).

Estimation of Bacillus amyloliquefaciens, and

Glomus intraradices, and NaCl on germination

and seedling growth of the superior variety

526 of Sorghum spp

As a second experiment, variety 526 was carried

out to evaluate the effect of temperature on

germination alternating temperature regimes of

5-15 (night-day), 10-20, 15-25, 20-30, and 25-

35°C, based on a 24-hr cycle of 12 hr (Growth

chambers with Sylvania cool white fluorescent

lamps, 25 μmol m-2 s-1, 400-750 nM). Seeds

were submitted at the same treatment

(disinfected and inoculated with PGPB and AMF

treatments under NaCl solutions (0, 0.06, and

0.12 M) as it was indicated in the first

experiment. Variables as percentage

germination, germination rates, root length,

height, and dry and fresh weights were evaluated

as previously cited in the first phase. All data

were analyzed using the Statistical Analysis

System (Maguire 1962; SAS 2004). A hierarchic

experiment of randomized was designed with

three factors (inoculation, temperature regime,

and salinity); same three levels as a 1st

experiment were analyzed: a) without inoculants,

with inoculant PGPB (B. amyloliquefaciens), and

AMF (Glomus intraradices); three

concentrations of NaCl (0, 0.06, and 0.12 M), and

c: five temperature regimes (5-15, 10-20, 15-25,

20-30, and 25-35 °C, based on a 24-hr cycle of

12 hr).

RESULTS

Germination and seedling growth of four

Sorghum varieties

The germination percentage was significantly

modified by biotreatments studied in this

experiment (B. amyloliquefaciens and Glomus

intraradices), under salinity conditions (Table I).

When salinity was higher (0.12 M NaCl),

inhibition of germination was observed in all

varieties non inoculated (< 80%), compared with

those inoculated, sticking out the 526 variety

with B. amyloliquefaciens. However, when

salinity conditions were reduced at 0 M NaCl),

germination percentage was increasing until 97%

in 526 Var. + with both beneficial

microorganisms. The contrary was observed in

varieties non biotreated, obtaining significant

germination results in 76% at 0 M, and obtaining

high values the 526 var. Similar behavior was

showed in 0.06 M of NaCl, where 526 var.,

showed the highest values in germination with

and even without Plant Growth promoting

Bacteria (PGPB) and arbuscular mycorrhizal

fungi (AMF). According to the germination rate,

results (data no-showed) of all varieties with bio-

inoculants studied, showed a total germination

(after inoculation and sowed – AIS-), among 8 to

17 days =d= (at 0 M NaCl = 9d with a 99.50%;

at 0.06 M = 11d with 55%, and 0.12 M NaCl =

17 d with no more than 43%), while those non

inoculated showed germination total rate till

11dAIS to 20d; at 0M with a 82.35%; at 0.06

with 43% in 16d AIS, and no more than 42% at

0.12 M NaCl in 20d, respectively. PGPB and

AMF influenced plant growth of all varieties,

during early seedling with significant differences

among treatments for plant height, root length,

fresh and dry weight (Table I). These results

evidenced the potential on 526 var. to be selected

and used in the next step. All high values were

obtained at 0 M of NaCl with B.

amyloliquefaciens and Glomus intraradices, and

highlighting the same bionoculants at 0.06 and

0.12 M vs treatments without beneficial

microorganisms.

Evaluation of B. amyloliquefaciens and

Glomus intraradices inoculants, NaCl levels,

and temperature regimes on germination and

seedling growth of 526 variety

After inoculation with PGPB and AM during the

germination stage under NaCl levels and

temperature regimes, results showed significant

differences among treatments (Table II). Optimal

germination of 526 var. in 12/12 hr light/dark

conditions occurred under all salinity conditions,

were obtained at 0 M NaCl (P>0.05), and in these

all treatments whit bio inoculants (P>0.05), but

no significant between temperatures 5/15, 10/20,

15/25, and 25/35 °C (Table II).

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Table I Effects of Bacillus amyloliquefaciens and Glomus intraradices on average values of germination (%), root length, plant height, fresh and

dry weights of seedlings of varieties of Sorghum spp (Silo Miel 370 VCB (370), Sorgo Silo Miel 315 VC (315), Sorgo Silo Miel II (II) and Camino

526 CROPLAM WIN FIELD (526)) under three concentrations of NaCl after 25 days sowed.

Variety Inoculant* NaCl concentration

(M)

Germination

(%)

Plant height

(cm)

Root

length

(cm)

Fresh

weight

(g)

dry weight

(g)

370 Control 0 61.38d 10.20bc 11.60b 39.929a 7.591a

315 Control 0 79.64bc 11.40bc 11.15b 36.014ab 6.794a

II Control 0 78.53bc 10.90bc 9.05bc 35.927ab 7.597a

526 Control 0 87.21b 11.65bc 12.10b 40.911a 8.581a

370 B.amyloliquefaciens 0 83.44b 12.25bc 19.25a 40.914a 10.584a

315 B.amyloliquefaciens 0 86.65b 15.85b 15.70ab 38.927a 8.597a

II B.amyloliquefaciens 0 87.43b 17.95b 15.60ab 42.021a 10.801a

526 B.amyloliquefaciens 0 97.99a 24.80a 20.05a 43.924a 10.594a

370 G.intraradices 0 83.12b 13.20bc 15.65ab 39.917a 9.587a

315 G.intraradices 0 85.52b 12.55bc 16.60a 37.811a 7.481a

II G.intraradices 0 84.31b 15.35b 14.55ab 41.014a 10.684a

526 G.intraradices 0 96.90a 24.25a 20.25a 43.817a 10.487a

370 Control 0.06 34.22g 5.30d 4.25d 11.047c 4.387b

315 Control 0.06 32.56g 4.65d 5.55cd 10.063c 4.519b

II Control 0.06 37.78g 4.85d 5.30cd 11.066c 4.406b

526 Control 0.06 41.71f 5.40d 5.45cd 11.057c 4.397b

370 B.amyloliquefaciens 0.06 49.12e 9.05cd 7.70cd 12.743c 4.979b

315 B.amyloliquefaciens 0.06 48.21e 8.90cd 7.85cd 12.066c 4.269b

II B.amyloliquefaciens 0.06 42.12f 8.70cd 7.70cd 12.147c 4.777b

526 B.amyloliquefaciens 0.06 51.44e 13.90b 8.60cd 13.743c 5.979b

370 G.intraradices 0.06 48.56e 6.00d 6.85cd 11.066c 5.569b

315 G.intraradices 0.06 48.22e 8.45cd 7.05cd 11.947c 4.777b

II G.intraradices 0.06 44.32ef 7.30d 7.20cd 10.853c 4.199b

526 G.intraradices 0.06 50.70e 12.80bc 7.80cd 12.846c 4.076b

370 Control 0.12 15.21j 1.35ef 1.10e 7.740cd 0.904bc

315 Control 0.12 13.56j 1.65ef 1.50e 7.033cd 1.082bc

II Control 0.12 13.12j 1.95ef 1.50e 7.049cd 1.518bc

526 Control 0.12 17.32j 1.55ef 1.25e 7.510cd 0.904bc

370 B.amyloliquefaciens 0.12 26.11h 3.10de 6.15cd 7.036cd 1.062bc

315 B.amyloliquefaciens 0.12 21.98i 3.90de 6.90cd 8.719c 1.018bc

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Variety Inoculant* NaCl concentration

(M)

Germination

(%)

Plant height

(cm)

Root

length

(cm)

Fresh

weight

(g)

dry weight

(g)

II B.amyloliquefaciens 0.12 29.11gh 2.55de 6.25cd 8.730c 0.944bc

526 B.amyloliquefaciens 0.12 29.23gh 3.95de 8.80bc 9.366c 1.062bc

370 G.intraradices 0.12 27.21h 3.85de 5.65cd 9.269c 1.018bc

315 G.intraradices 0.12 23.14hi 3.00de 5.55cd 8.730c 0.944bc

II G.intraradices 0.12 28.22gh 3.15de 6.60cd 8.476c 1.082bc

526 G.intraradices 0.12 28.57gh 3.95de 8.70bc 9.709c 1.018bc

Means followed by the same letter are not significantly different at P = 0.05. Comparisons were made within columns using Duncan’s Multiple Range Test. Values are means

of five replicates. *Bacteria (1x108 CFU mL-1); AMF (120 propagules.cm3). control=C without inoculants.

Table II. Effects of Bacillus amyloliquefaciens and Glomus intraradices on average values of germination (%), root length, plant height, fresh and dry weights of

seedlings in 526 Var. of Sorghum spp., under three concentrations of NaCl at temperatures of 5-15, 10-20, 15-25, 20.30 and 25-35°C.

Variety Inoculant*

Temperature

regimens

°C

NaCl

concentration

(M)

Germination

(%)

Plant

Height

(cm)

Root

length

(cm)

Fresh

weight

(g)

Dry

weight

(g)

526 Control 5/15 0 77.05b 11.42b 16.82c 62.241b 6.882a

526 B.amyloliquefaciens 5/15 0 82.35ab 14.57b 18.77c 80.244a 8.901a

526 G.intraradices 5/15 0 82.00ab 14.02b 16.97c 79.247a 8.811a

526 Control 5/15 0.06 39.05d 5.17d 7.17e 14.267cd 2.703b

526 B.amyloliquefaciens 5/15 0.06 52.25c 8.67d 8.32e 20.283c 3.094b

526 G.intraradices 5/15 0.06 51.85c 8.57d 8.52e 22.276c 3.115b

526 Control 5/15 0.12 18.65e 2.32de 2.97ef 14.280cd 0.604c

526 B.amyloliquefaciens 5/15 0.12 42.05d 8.72d 5.52e 19.246c 0.768c

526 G.intraradices 5/15 0.12 41.65d 6.72d 5.42e 18.259c 0.751c

526 Control 10/20 0 78.00b 9.42d 15.51c 63.141b 7.012a

526 B.amyloliquefaciens 10/20 0 83.30ab 14.42b 19.82c 81.132a 8.911a

526 G.intraradices 10/20 0 82.95ab 13.22b 18.22c 80.238a 8.281a

526 Control 10/20 0.06 40.00d 6.72d 6.29e 15.137cd 2.453b

526 B.amyloliquefaciens 10/20 0.06 53.20c 8.87d 9.79e 21.273c 3.124b

526 G.intraradices 10/20 0.06 52.80c 7.37d 10e 23.276c 3.215b

526 Control 10/20 0.12 19.60e 3.97de 3.09e 15.260cd 0.658c

526 B.amyloliquefaciens 10/20 0.12 43.00d 7.82d 7.55e 20.226c 0.759c

526 G.intraradices 10/20 0.12 42.60d 7.49d 7.57e 19.239c 0.793c

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Variety Inoculant*

Temperature

regimens

°C

NaCl

concentration

(M)

Germination

(%)

Plant

Height

(cm)

Root

length

(cm)

Fresh

weight

(g)

Dry

weight

(g)

526 Control 15/25 0 85.20a 15.82bc 15.51c 65.361b 7.342a

526 B.amyloliquefaciens 15/25 0 99.50a 26.97a 30.05a 83.244a 8.341a

526 G.intraradices 15/25 0 99.15a 26.42a 28.47a 83.115a 8.341a

526 Control 15/25 0.06 40.20d 8.57d 6.18e 16.237cd 2.673b

526 B.amyloliquefaciens 15/25 0.06 53.40c 15.07b 14.85c 22.263c 3.784b

526 G.intraradices 15/25 0.06 53.00c 14.97b 15.05c 22.146c 3.215b

526 Control 15/25 0.12 19.80e 1.72e 1.96ef 16.260cd 0.638c

526 B.amyloliquefaciens 15/25 0.12 43.20d 5.12d 12.05cd 21.776c 0.732c

526 G.intraradices 15/25 0.12 42.80d 3.12de 11.96cd 20.479c 0.716c

526 Control 20/30 0 78.20bc 19.82ab 15.83c 64.131b 6.982a

526 B.amyloliquefaciens 20/30 0 83.50ab 25.97a 25.29ab 82.142a 8.841a

526 G.intraradices 20/30 0 83.15ab 25.42a 23.5b 82.338a 8.921a

526 Control 20/30 0.06 43.20d 9.57d 5.85e 17.137c 2.433b

526 B.amyloliquefaciens 20/30 0.06 56.40c 18.07b 19.72c 24.033c 3.334b

526 G.intraradices 20/30 0.06 56.00c 17.97b 19.59c 24.036c 3.235b

526 Control 20/30 0.12 22.80e 2.72de 1.76ef 17.270c 0.615c

526 B.amyloliquefaciens 20/30 0.12 46.20d 7.12d 16.82c 22.686c 0.769c

526 G.intraradices 20/30 0.12 45.80d 5.12d 16.59c 21.699 0.693c

526 Control 25/35 0 79.20b 14.32bc 15.5c 64.831b 6.222a

526 B.amyloliquefaciens 25/35 0 84.50ab 25.47a 33.29a 82.742a 8.991a

526 G.intraradices 25/35 0 84.15ab 24.92a 31.49a 82.128a 8.111a

526 Control 25/35 0.06 41.20d 8.07d 6.19e 16.327cd 2.213b

526 B.amyloliquefaciens 25/35 0.06 54.40c 16.57b 22.5b 22.363c 3.334b

526 G.intraradices 25/35 0.06 54.00c 16.47b 22.82b 22.956c 3.785b

526 Control 25/35 0.12 20.80e 1.22de 4.54e 16.380cd 0.670c

526 B.amyloliquefaciens 25/35 0.12 44.20d 5.62d 19.82c 21.126c 0.774c

526 G.intraradices 25/35 0.12 43.80d 3.62de 19.83c 20.139c 0.774c

Means followed by the same letter are not significantly different at P = 0.05. Comparisons were made within columns using Duncan’s Multiple Range Test. Values are means

of five replicates. *Bacteria (1x108 CFU mL-1); AMF (120 propagules.cm3). control=C without inoculants.

Tropical and Subtropical Agroecosystems 23 (2020): #48 Holguin-Peña et al., 2020

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Table III. Results of two way ANOVA carried out on factors of salinity, thermoperiod,

microorganisms and their interactions.

Independent variable Salinity Thermoperiod microorganisms Salinity*Thermoperiod

Percent germination 0.000000 0.000000 0.000000 0.6419056

High plant 0.000000 0.000000 0.016424 0.224755

Root length 0.153204 0.000000 0.000000 1.000000

Fresch weigh 0.000000 0.839958 0.000000 0.977109

Dry weight 0.000000 1.000000 0.000001 1.000000

Note: Numbers are F-values, P = 0.05

Continuation….

Table III. Results of two way ANOVA carried out on factors of salinity, thermoperiod,

microorganisms and their interactions.

Independent

variable

microorganisms*

Salinity

microorganisms*

Thermoperiod

microorganisms*Thermoperiod*

salinity

Percent

germination 0.059789 0.138434 1.000000

High plant 0.035483 0.923315 0.833949

Root length 0.003487 0.000000 1.000000

Fresch weigh 0.000000 0.998041 0.969960

Dry weight 0.000000 1.000000 1.000000

Note: Numbers are F-values, P = 0.05

However, was detected that numerically among

treatments inoculated with the bacterium PGP

and AMF, stood out B. amyloliquefaciens at

20/30 °C, with a minimum difference compared

with Glomus intraradices (99.50 vs 99.15,

respectively), while non-inoculated treatment, it

showed 85.30%. According to with plant height

variable, highest values were obtained with 0 M

NaCl, with both beneficial inoculants compared

with control non-bioinoculated and at all

temperatures excepted 5/15 °C. Similar behavior

was displayed in root length, fresh and dry

weight variables. Different inoculants,

temperatures and several concentrations of

salinity individually, and their interaction,

significantly affected the rate of germination of

526 variety seeds (Table III).

DISCUSSION

Sorghum acts as a dietary staple for millions of

people living in about 30 countries in the

subtropical and semi-arid regions of Africa, Asia,

and Mexico (Rakshit et al. 2014; Hariprasanna

and Rakshit 2016). It is a source of food and

fodder, mostly in the traditional, smallholder

farming sector. Maunder (Maunder 1999), and

Juarez and Harrison (20), indicate that in dry arid

zones, more than 80 % of the global Sorghum

area is characterized by low yield levels due to

the low content of organic matter, and in this

sense, farmers should apply many chemical

fertilizers to supply N deficiencies. To overcome

the challenge of increasing food production with

a significant reduction of agrochemical use and

environmental pollution, and an increase of

natural resource productivity, the use of soil

microorganisms in basic crops like Sorghum is

essential. Two groups of microorganisms consist

of plant growth-promoting bacteria (PGPB) and

arbuscular mycorrhizal fungi (AMF), which have

been studied from the beginning of the twentieth

century and their mode of action at the

physiological level is currently well understood

(Bashan 1998; Vessey 2003; Ruzzi and Aroca

1980).

Sorghum spp. includes different varieties which

are sowed along of northwest of México in two

seasons each year. Sorghum seeds are exposed to

two different environments at planting seasons;

from May to June, the temperature exceeds 47

°C, and the relative humidity does not exceed

5%; so the water potentials inside of halotolerant

plants (sap) and in soil show values between 7

and 8 –Mpa, respectively; this phenomenon

decreases until September and October below 5

and 4 -Mpa in gross sap and soil respectively.

Therefore, this abiotic stress present in desert

areas must be reduced and strengthened by the

interaction of beneficial microorganisms as our

study showed and with the variety "526" (Table

I, II). Studies related to interaction plant and

beneficial microorganisms agree with our

findings (Baldani and Dobereiner 1980; Bashan

et al. 2014; Bashan et al. 2012), who indicated

that results depend on strain-host plant

specificity. Specifically with Sorghum studies,

Tropical and Subtropical Agroecosystems 23 (2020): #48 Holguin-Peña et al., 2020

9

our results are strengthened with those found by

Sarig et al. (Sarig et al. 1992), Issopi et al.

(1995), James et al. (1997), Stein et al. (1997),

they obtained favourable results with the

inoculation of beneficial microorganisms like a

vesicular-arbuscular mycorrhizas and

Acetobacter diazotrophicus; Herbaspirillum, as

a endophytic diazotroph colonizing vascular

tissue in leaves; Azospirillum brasilense

inoculation on growth dynamics and hydraulic

conductivity; and the contribution of nitrogen

fixation by Azoarcus sp. BH72 in Sorghum,

respectively. However, disagree with Arsac et al.

(1990), Okon and Labandera (1994), who stated

that the effect is not strain-dependent among

different plant species.

Our study shows that low temperatures reduce

germination; the contrary was observed in

germination percentage with an increase in

temperature (Table 2). For its part, while the

salinity was reduced, the germination was

increased. At 15 °C and under 0.12 M NaCl, our

526 var. germinated and showed tolerance to

salinity; however the germination of this seed

was more effective when we inoculated PGPB;

results agreeing with others results in others

crops (Khan 1991); Khan and Weber (1986),

reported maximum salinity tolerance ranges

from 200 to 1720 mM NaCl in halophytes such

as S. bigelovii, S. europaea, S. stricta, Cressa

cretica, and Suaeda moquinii. These reports

indicate that 526 variety could be classified into

the salt-tolerant one during germination.

In this 2nd phase, the temperature increasing

germination percentage in 2 and 13% at 20/30

and 15/25, respectively, compared with the 1st

phase using 27 °C in a constant form (at 0 M

NaCl and both bio-inoculants). Moreover, the

same variety in the other variables (plant height,

root length, fresh and dry weight variables), its

values were significant vs control non inoculated

when seeds were inoculated with PGPB and

AMF. Considering a relationship between all the

variables with the different temperatures and

salinity studied, some plants showed variability

in their length and weight in their roots and whole

plant. This is attributed to the capacity of the

plant to tolerate in its interior ions and the

production of glycophytes and osmolytes, which

are have a function of maintaining an ionic

homeostasis between several tissues and

intracellular compartmentalization; effects

visualized in desert species of the Great Basin,

subtropical maritime deserts of Pakistan,

Haloxylon recurvum, Zygophyllum simplex and

Arthrocnemum macrostachyum (Khan and

Ungar 1997; Khan 1991; Gul, B.; Weber 1999),

respectively.

All varieties studied in this study and especially

“526” obtained, was benefit using beneficial

microorganisms by B. amyloliquefaciens and G.

intraradices abilities. Other studies indicate

positive effects using plant growth-promoting

microorganisms (Afek et al. 1990), results that

coincide with those obtained in the present study,

appreciating that the PGPB of this study and

experiencing it with var. 526 Sorghum, positively

was affected. These effects are possible to the

participation of responsible substances in the

growth of plants (Afek et al. 1990; Puente

Bashan 1993; Goodfriend et al. 2000; Díaz et al.

2001). Similarly, some studies support G.

intraradices in the promotion of plant

development (Melgares et al. 2004), results that

are consistent with the findings in this evaluation.

Similar results were obtained for other plants and

beneficial microorganisms (90). However, other

assays although they were carried out with other

plants and other beneficial microorganisms, also

some inhibitive effects on germination were

observed (Díaz et al. 2001).

Plants exposed to saline stress change their

environment, and one of the first vegetative

phases is germination. In this first stage, osmotic

stress begins to manifest itself, mainly by

stopping the emergence of the radicle and its

subsequent roots. The recoil is associated with an

accumulation of ionic stress that generates

phytotoxicity, and showing an imbalance in

nutrient intake and absorption (Asish and Anath

2005), oxidative stress at the subcellular level,

mediated by reactive oxygen species (ROS)

(Acosta-Motos et al. 2017), mainly. These

responses to salinity in plants tolerant to NaCl,

implement adaptations so as not to be affected by

salinity and carry out their survival. There are

mechanisms inside the cells that promote an

adaptation that is reflected in their morphology,

physiology, biochemistry and molecular

(Acosta-Motos et al. 2017). In the germination

stage, among the first existing metabolic actions

to tolerate salt, it is determined by multiple routes

that influence in water retention and / or

acquisition, protecting organelles and

maintaining homeostasis. Given this situation,

that the seeds or new plants are subject, the

beneficial microorganisms as a PGPB or AMF,

release bio substances that generate hormonal

changes within the plants, the production of

volatile organic, benefiting in the taking of

nutrients and a tolerance to abiotic stress such as

salinity (Ruzzi and Aroca 2015). Same authors

indicate that oxidative stress in different phases

in the plant is linked to the electron transport

chain in membranes of mitochondria and

chloroplast during ATP production. Therefore,

Tropical and Subtropical Agroecosystems 23 (2020): #48 Holguin-Peña et al., 2020

10

numerous energetically demanding

physiological processes like germination and

seedling development are associated with

oxidative stress (Bailly 2004). It is well known

that mitochondria are the principal subcellular

compartment of oxygen consumption and the

principal source of reactive oxygen species

(ROS) (Puntarulo et al. 1998). Most of the ATP

required for early stages of development comes

from mitochondria; therefore, generation of ROS

during this process is mainly associated to

cellular respiration. At later stages of

development, photophosphorylation in

chloroplast becomes the main source of ATP to

cover the energy requirement for proper

development; then, chloroplast also becomes a

primary source of ROS (Asada 2006), and it is

possible that the release of hormones by

beneficial microorganisms inside and attached to

plants, the production of volatile organic

compounds, help to improve nutrient intake and

improve tolerance to abiotic stress (García et al.

2018).

CONCLUSIONS

This study is the first step to obtain an ideal

variety of Sorghum, which is a principal crop in

dry arid and desert zones of México. This kind of

study promotes beneficial microorganisms as a

resource to increase the development and

productivity of plants. Moreover, studies with the

interaction of B. amyloliquefaciens and G.

intraradices with 526 var. of Sorghum are

necessary to decide the magnitude to which these

observations can be observed and reproduced

under one production system.

Acknowledgments Thanks to personal technicians of Laboratories in

CIBNOR. In memorial of Dr. Rogelio Ramirez

SerranoT at CIBNOR, QEPD.

Funding. We thank to Universidad de Sonora by

Project “Effect of promoting halobacteries of

vegetable growth in the ecophysiology and

production of the Sorghum crop under saline

conditions”, key: USO313006248.

Conflict of interest. The authors declared no

conflict of interest.

Data availability. Data are available with the

corresponding author

(erueda04@santana.uson.mx) upon reasonable

request

Compliance with ethical standards. There was

strict adherence to the Global code of conduct for

research in resource-poor settings following the

Convention on Biological Diversity and

Declaration of Helsinki.

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